This proposal examines the mechanism for the altered collagen gene expression after transformation of cells to tumor cells. Collagen, an important connective tissue component, plays a significant role influencing cell behavior and maintaining tissue structure. It has been established that collagen gene expression usually decreases when cells become transformed which may have significance to tumor growth. Establishing the mechanism controlling collagen synthesis may lead to understanding the role of collagen in tumor growth. Since human tumors are usually carcinomas of epithelial origin, we have examined several epithelial-like cell lines. The model system used in this proposal is a carcinogen, 2-N- (acetoxyacetyl) amino fluorine (AAF), transformed rat liver epithelial-like cell line, W8, derived from K16 cells. The collagen produced by the W8 cells is an alpha(1) trimer with no alpha2(I) chains due to inhibition of alpha2(I) mRNA transcription. Unlike virally infected transformed fibroblasts, the W8 cells have normal trans-acting factors as judged by transfection studies. Therefore, transcription of alpha2(I) is probably inhibited by an alteration in the collagen promoter or enhancer. There is evidence that the W8 alpha2(1) promoter is methylated whereas other alpha2(I) promoters are not methylated. This alteration may be involved in inhibition of transcription. Therefore, our W8 and K16 cells provide a unique model system to locate critical areas in the promoter that regulate collagen gene expression. Our hypothesis is that the W8 cells have normal trans-acting factors that are non-functional because of alterations in the promoter (and/or enhancer) region of the alpha2(1) gene. During this grant period, the regulatory regions of the alpha2(1) gene will be isolated from W8 and normal gene libraries in order to locate methylations and/or mutations in the W8 alpha2(1) gene. The activity of these regions with and without methylations and/or mutations will be tested in transfection assays. Experiments are planned to examine the significance of methylation. Once the critical regions for promoter activity are determined, than we will begin to examine the DNA binding proteins and transcriptional enhancement proteins that might interact with these areas.